Occurrence of Myxobolus spp. (Myxozoa) in the blood of Metynnis lippincottianus (Osteichthyes: Serrasalmidae) from eastern Amazon, Brazil

Abstract Myxozoans are obligatory parasites and can be found in various organs and bloodstreams of fish, thus, the objective of this work was to describe the occurrence of Myxobolus spp. in the circulating blood of Metynnis lippincottianus from River Curiaú, Macapá City, eastern Amazon, Brazil. The samples of M. lippincottianus (11) were caught using cast net and gillnets. The fish blood was collected by puncturing the caudal vessel, using needles and syringes containing 10% of EDTA solution. Blood smear were prepared and panchromatic stained with a combination of May Grunwald-Giemsa-Wright, for observation and examination of the parasitic structures in optical microscope. Tissues from the kidney was examined using specific stereoscopic binoculars to check for the presence of cysts, lesions and parasites. The prevalence of Myxobolus spp. infecting the circulating blood of the fish was 36.36% (4/11) and 15 spores of mixosporyds were visualized. Myxobolus spp. had a prevalence of 54.55% (6/11) in host's kidney tissue and the morphometric spores data converge with observed in the blood. The morphological characteristics of the spores in the blood samples revealed two morphotypes of Myxobolus spp. This is the sixth occurrence recorded of Myxobolus spp. infecting fish blood in Brazil.


Introduction
Parasitism in fish is a common phenomenon in natural and farmed aquatic environments, where various species of parasites can infect and affect the health of their hosts.These parasites can be found in various parts of the fish body, such as the gills, muscles, skin, intestines, liver, and blood, where they can cause various diseases.Some parasites can infect a wide range of hosts whereas others are specific to both the species of fish and the body tissues infected (Molnár, 2002;Sitjà-Bobadilla, 2008;Ferreira et al., 2021).

Metynnis lippincottianus
Cope, 1870, is a freshwater pelagic fish, with a wide distribution in South American water systems, including the drainages of French Guiana, the basins of the lower and middle Amazon and the drainages of northeastern Brazil (Zarske & Géry, 1999;Froese & Pauly, 2023).Popularly known as "pratinha", "pacu," and "pacuredondo", M. lippincottianus has a generalist feeding behavior, with a diet based on filamentous algae, terrestrial and aquatic plants, detritus, microcrustaceans and ostracod molluscs (Hoshino & Tavares-Dias, 2014;Kliemann et al., 2022), and it belongs to the Serrasalmidae family, which is important in the Brazilian aquarium trade as ornamental fish, as well as part of the diet of riverside populations in the Amazon region (Moreira et al., 2009;Yamada et al., 2012).
Fish like M. lippincottianus can host distinct species of parasites, such as the Myxobolus genus, which has the greatest number of species.Some species of this genus are known to be pathogenic, causing specific diseases in fry of regional and exotic fish species, including "whirling disease" in salmonids and myxobolosis in certain round fish (Steinbach Elwell et al., 2009).The clinical signs can vary according to the fish species host and the location of infection (Békési et al., 2002;Oliveira et al., 2021;Capodifoglio et al., 2019).
This type of infection can cause various problems for the host fish, and the clinical signs can compromise their health and well-being.Another associated problem is parasite transmission to other fish species or even adaptation to new infection sites.This can cause pathologies, lack of infection control, and spread of infection, increasing the risk of parasitic disease outbreaks in fish populations, causing greater problems and even an imbalance in hostlinked biodiversity (Santos et al., 2013;Farias et al., 2021).Thus, fish parasitology is an important area of study for understanding the epidemiology of parasitic infections in fish, as well as for developing strategies to prevent and control parasitic diseases in aquatic environments.
In Brazil, studies about the occurrence of myxozoans infecting the blood of fish have been carried out in several regions (Central-West and North), with spores (myxospore phase) of the genus Myxobolus being the most common (Paperna & Di Cave, 2001;Maciel et al., 2011;Úngari et al., 2021).Also, Sphaerospora sp. were identified as parasitizing Amazonian fish.In Europe and Asia, other parasites of the genus Chloromyxum, Myxobolus and Sphaerospora have been detected in the kidney and blood of fish (Yang et al., 2022;Holzer et al., 2006;Baska & Molnar, 1988;Lom et al., 1985).
In order to contribute to information on hemoparasitism linked Myxobolus species in Amazonian fish, this study aimed to describe the occurrence of two morphotypes of this parasite in the circulating blood of M. lippincottianus (Osteichthyes: Serrasalmidae) from the Curiaú River, Macapá, Amapá, Eastern Amazon, Brazil.

Collection and transportation of biological material
Metynnis lippincottianus (n = 11) specimens were collected from the Curiaú River Environmental Protection Area (APA do Rio Curiaú) (0°8'43.6"N, 51°2'30.3"W) (Figure 1).The Curiaú River APA covers the Curiaú River basin, which has an area of approximately 584.47 km 2 , a drainage system interconnected by intermittent and perennial lakes, and the influence of the tidal and rainfall regime (Brito et al., 2022).
The fish were caught using cast net and gillnets with varying mesh sizes, to obtain a significant sample of individuals with different sizes and body weights.The fish were placed in plastic bags containing water from their own habitat, aerated using battery-operated aerators.The animals were then transported alive to the Laboratory of Morphophysiology and Animal Health -LABMORSA / UEAP, Macapá, AP.

Parasitological analysis
In the laboratory, the animals were kept in aquariums with constant aeration until further analyses.A blood sample was collected from each specimen by puncturing the caudal vessel using needles and syringes containing 10% EDTA.Blood smears and panchromatic staining with May Grünwald-Giemsa-Wright (Ranzani-Paiva et al., 2013) were performed to observe the parasite structures.The samples were examined for hemoparasites under an optical microscope (Lumen LM3100) at 40x and 100x magnification and identified as described by Lom & Dyková (2006).
The fishes were anesthetized using tricaine methanesulfonate (MS-222), followed by euthanasia with neural myelotomy, necropsy and macroscopic observation of the kidney tissues, to check for the presence of cysts, lesions and parasites.During the necropsy, kidney fragments were visualized fresh between slides and coverslips, tissues with cysts were collected and analyzed by optical microscopy.
Parasite spores were recorded using a Moticam 2300 3.0 M camera with Motic images Plus 2.0 software attached to the microscope.The morphometric data of mature and fresh spores were obtained (μm) according to Matos et al. (2001) and analyzed as recommended by Lom & Arthur (1989).

Results and Discussion
The M. lippincottianus specimens were 7.94 ± 0.54 cm in total length and 11.18 ± 1.54 g in body weight.The prevalence of Myxobolus spp.infecting the circulating blood of the fish collected in the Curiaú River was 36.36%(4/11) and 15 hemoparasites were found.Myxobolus spp.had a prevalence of 54.55% (6/11) in host's kidney tissue.However, clinical signs of infection, common to the genus, could not be observed in the parasitized fish.
Metynnis lippincottianus is a freshwater pelagic fish with a diversity of ectoparasites and endoparasites (Hoshino & Tavares-Dias, 2014), such as those of the subphylum Myxozoa.Myxobolus is a genus of the family Myxobolidae, belonging to the phylum Cnidaria and class Myxosporea, which has the largest number of members, with more than 900 species described in literature (Eiras et al., 2021).In this study, the myxospores had two pyriform polar capsules of equal size, bilateral symmetry, and binucleate sporoplasm, indicating that the identified parasites belonged to the genus Myxobolus (Figure 2).
The morphometric survey revealed two morphotypes of Myxobolus spp., one elongated in the shape of a drop (M1) and the other oval (M2) (Figure 3).The M1 parasites measured 17.00 ± 1.49 µm in spore length and 6.00 ± 0.61 µm in width; the polar capsules present the same size with 15.00 ± 1.13 µm long and 2.40 ± 0.37 µm wide, with 16 to 18 turns of the polar filament (Figure 3A).The M2 spores were on average 11.01 ± 0.07 µm long and 4.08 ± 0.30 µm wide; the polar capsules present the same size and with 6.53 ± 0.41 µm long and 1.56 ± 0.21 µm wide, with 4 to 6 turns of the polar filament (Figure 3B).
The drop-shaped spores showed morphological characteristics similar to those of the Myxobolus maculatus found parasitizing the kidney of the Amazonian fish, Metynnis maculatus (Casal et al., 2002) (Table 1).In this study, we could not determine the species level of Myxobolus spp.because, in addition to methods based on morphology, morphometry, and biology (location in host tissue, morphology of sporogonic stages, and other developmental stages), use of precision devices, such as molecular tools was needed (Holzer et al., 2010;Fujimoto et al., 2013).This group of parasites has an indirect and complex life cycle for two hosts, invertebrates, such as aquatic annelids, and intermediate vertebrates, mainly fish (Eszterbauer et al., 2015).Generally, the species of genus Myxobolus are morphologically characterized by their pyriform shape and presence of spores in their life cycle; they can be differentiated by the size of their polar capsules, number of polar filament turns, sporoplasm formation and valve formation (Lom & Arthur 1989;Fiala et al., 2015;De Araujo et al., 2018).
Further, organisms of the genus Myxobolus have two polar capsules, which originate from cells found inside the spore.The polar filaments have a helical shape and sporoplasm with a binucleate cell; the number of turns of the polar filament is important for characterizing and identifying the species.This cell contains several electrodense vesicles known as sporoplasmosomes (Casal et al., 2006).
In the Brazilian Amazon, the presence of myxosporidians has been recorded in M. lippincottianus, which the presence of Henneguya sp. has been reported and two morphotypes of Myxobolus sp. in the gills of this host (Carvalho et al., 2019).In work carried out by Carvalho et al. (2020), the presence of Henneguya sp. in 80% of the gills of M. lippincottianus caused hyperplasia and fusion of the gill lamellae.Both studies were carried out in the same collection area as this research.
In Brazil, only five records of Myxobolus spp.were reported in the fish blood (Table 2), and this work is the first record of the occurrence of Myxobolus spp.parasitizing the blood of M. lippincottianus.It is worth mentioning that this species of myxozoan has already been found in several fish organs, such as gills, blood vessels and caudal kidney (Cirkovic et al., 2010;Mabika et al., 2016;Manrique et al., 2017;Batueva, 2020;Maftuch et al., 2021;Silva et al., 2023), with the bloodstream being the least recurrent organ for Myxobolus sp.parasitism, which demonstrates the degree of relevance of this work.

Myxobolus
According to Holzer et al. (2006), the presence of myxosporeans may be related to the use of blood as a means of transportation and a channel for parasite proliferation in the target organs, the kidney and the gall bladder, thus demonstrating a possible justification for the evidence obtained in this study.Maciel et al. (2011), state that blood samples from fish must be evaluated to report the presence of myxozoans, since in their study the possibility of blood contamination due to the presence of this parasite, whether in the form of plasmodium, free spores in the mucosa, epithelium or connective tissues that were accessed during the puncture of the caudal vein.
In this study, spores of Myxobolus spp.were identified in the fresh caudal kidney, and the morphometric data of the spores found in the blood converge with those observed in the kidney.The spores of Myxobolus spp.develop in the internal organs from where mature myxospores are transported via the bloodstream to reach the target organ, caudal kidney (Molnár et al., 2009;Bjork &Bartholomew, 2010 andManrique et al., 2017).The observed results reinforce the hypothesis that Myxobolus spp.complete their sporogonic development or with release into the environment from the host urine.Sipos et al. (2018) proved through experimental results that Myxobolus cerebralis is found in blood during the initial stage of host infection, and that the intensity of infection in the blood decreases over time and with the growth phase.
Detailed studies on the morphology of Myxobolus spp.are important for understanding the biology and epidemiology of these parasites, as well as for developing effective diagnostic and treatment methods in cases of parasite outbreaks.Further, the morphology of spores, polar capsules and the number of valves can provide valuable information for identifying and characterizing different Myxobolus species that affect fish in the Amazon region.
The results obtained and compared with the literature demonstrated in this study allowed us to conclude that Myxobolus spp.are parasites of M. lippincottianus (Osteichthyes: Serrasalmidae) originating from the Curiaú River and although the fishes analyzed were apparently healthy, studies like this are sound, they are essential to protect the health and survival of fish populations and to maintain the health of aquatic ecosystems as a whole.So, considering the relevance and importance of this study, further research is needed to obtain more information on the life cycle in this intermediate host and the epidemiological potential linked to the infection of Myxobolus spp. in the circulating blood and kidney of M. lippincottianus.

Figure 1 .
Figure 1.Brazil map with Amapá state, highlighting the Curiaú River Environmental Protection Area and its hydrography (gray line).The dot indicates the location where Metynnis lippincottianus specimens were captured.

Table 1 .
Comparative measurements of Myxobolus spp. in Metynnis lippincottianus with other serrasalmids species of Brazil.

Table 2 .
Occurrence of Myxozoa parasites in fish blood; Myxobolus sp1.: drop-shaped; Myxobolus sp2.: oval-shaped.Myxobolus spp.(Myxozoa)in the blood of Metynnis the Masters Degree scholarship, in which has been developing a key role for the expansion and consolidation of Science in Brazil.Special thanks to the University of the State of Amapá for the financial support given to research projects associated to groups (PROGRUPOS).